Methods and systems for matching product ions to precursor in tandem mass spectrometry

ABSTRACT

Methods of tandem mass spectrometry (MS/MS) for use in a mass spectrometer are disclosed, the methods characterized by the steps of: (a) providing a sample of precursor ions comprising a plurality of ion types, each ion type comprising a respective range of masses; (b) generating a mass spectrum of the precursor ions using the mass spectrometer so as to determine a respective mass value or mass value range for each of the precursor ion types; (c) estimating an elemental composition for each of the precursor ion types based on the mass value or mass value range determined for each respective ion type; (d) generating a sample of fragment ions comprising plurality of fragment ion types by fragmenting the plurality of precursor ion types within the mass spectrometer; (d) generating a mass spectrum of the fragment ion types so as to determine a respective mass value or mass value range for each respective fragment ion type; (e) estimating an elemental composition for each of the fragment ion types based on the mass value or mass value range determined for each respective fragment ion type; and (f) calculating a set of probability values for each precursor ion type, each probability value representing a probability that a respective fragment ion type or a respective pair of fragment ion types was derived from the precursor ion type. Some embodiments may include a step (g) of generating a synthetic MS/MS spectrum for each respective precursor ion type based on the calculated probability values.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the United States National Stage Application, under35 U.S.C. 371, of International Application PCT/US2010/032114 having aninternational filing date of Apr. 22, 2010, which claims the benefit ofthe filing date, under 35 U.S.C. 119(e), of U.S. Provisional Application61/176,812, filed on May 8, 2009.

TECHNICAL FIELD

This invention relates to methods of and systems for obtaining andanalyzing tandem mass spectrometry data.

BACKGROUND ART

Structural elucidation of ionized molecules of complex structure, suchas proteins is often carried out using a tandem mass spectrometer, wherea particular precursor ion is selected at the first stage of analysis orin the first mass analyzer (MS-1), the precursor ions are subjected tofragmentation (e.g., in a collision cell), and the resulting fragment(product) ions are transported for analysis in the second stage orsecond mass analyzer (MS-2). The method can be extended to providefragmentation of a selected fragment, and so on, with analysis of theresulting fragments for each generation. This is typically referred toan MS^(n) spectrometry, with n indicating the number of steps of massanalysis and the number of generations of ions. Accordingly, MS²corresponds to two stages of mass analysis with two generations of ionsanalyzed (precursor and products). A resulting product spectrum exhibitsa set of fragmentation peaks (a fragment set) which, in many instances,may be used as a fingerprint to derive structural information relatingto the parent peptide or protein.

Unfortunately, the above-described procedure of sequentially isolatingand fragmenting each precursor ion, in turn, may not provide greatenough throughput for analyses of complex mixtures of biomolecules. Foremerging high-throughput applications such as proteomics, it isimportant to provide as-yet unattainable speeds of analysis, on theorder of hundreds of MS/MS spectra per second. The throughput may beincreased by obtaining spectra containing a mixture of fragment sets (a“multiplexed” spectrum), the mixture produced by fragmenting multipleparent ions simultaneously, instead of sequentially. The finalmultiplexed spectrum contains products from a mixture of precursors, incontrast to an MS/MS spectrum in which the products come from a singleisolated precursor.

Procedures for obtaining and analyzing multiplexed spectra canpotentially reduce hardware complexity, since an upstream mass analyzermay be eliminated. Analysis of product ions produced by multipleprecursor ions can also better utilize the spectral bandwidth ofhigh-resolution mass analyzers, such as Fourier Transform Ion CyclotronResonance and Orbitrap mass spectrometers. However, interpretation ofthe potentially large number of fragment peaks in the resultingmultiplexed spectrum can be challenging.

Multiplexing is a general strategy for increasing throughput when thecapacity of a communication channel far exceeds what is required to sendan individual message at a specified fidelity. Under certain conditions,it may be possible to send multiple messages through the channelsimultaneously without appreciable information loss. In communicationsystems, the individual signals are encoded before being combined at thetransceiver to allow the detected signal to be “demultiplexed” orseparated into the original component signals at the receiver. The twomost common examples of multiplexing are time and frequencymultiplexing. In either case, the channel is partitioned into discretesub-channels.

In the field of mass analysis, the simultaneous measurement of multipleions by a Fourier transform mass spectrometer (e.g., LTQ-FT orLTQ-Orbitrap) is an example of frequency multiplexing. The signal fromeach ion populates a narrow band (of fixed width) in the frequencyspectrum of the Fourier-transformed transient signal. Typically, thesebands are distinct, i.e., non-overlapping, and can be triviallyseparated. In theory, the channel capacity of a Fourier-transform massspectrum is the ratio of the spectrum bandwidth divided by the bandwidthof an individual ion signal.

A Fourier transform mass spectrum has sufficient channel capacity toallow the simultaneous measurement of thousands of distinct ion masses,corresponding to neutral molecules present in a sample. However, the“code”, i.e., representing molecules by their masses, is degenerate,since multiple distinct molecules (e.g., isomers) can have identicalelemental compositions and therefore identical masses. Furthermore,molecules with masses that are distinct, but differ by less than thenominal mass accuracy, can be misidentified.

To overcome this limitation, additional information about the molecule'sidentity can be obtained, by breaking the molecule into fragments andmeasuring the masses of these product ions. The covalent structure of amolecule, which distinguishes it from its isomers, can be inferred froma sufficiently informative MS/MS spectrum and perhaps additional apriori information. Commercially available software products such asMASCOT and SEQUEST have been used to identify peptides by matching alist of masses extracted from such spectra to predicted product ionmasses generated from protein sequences stored in proteomic databases.These programs often provide correct identifications even when theproduct ions are measured with only unit mass accuracy and resolution.Unfortunately, in conventional practice, an entire spectrum is used tomeasure the product ions from one precursor. This represents a dramaticbottleneck in throughput.

The present invention takes advantage of the concept that the additionalinformation provided by high-mass-accuracy (e.g. 1 part-per-million(ppm) rather than unit mass accuracy) and high-resolving-powermeasurements of product ions can support mass-spectral de-multiplexing.Such de-multiplexing permits greater sample throughput. In other words,the availability of high-resolution and high-accuracy spectrometersmakes it possible, in certain instances, to identify multiple precursormolecules from a single high quality spectrum that contains a mixture ofproduct ions derived by fragmentation of these multiple precursors. Theadditional mass accuracy of the fragments can enable development ofalgorithms to discover the correct assignment of product ions toprecursors while also compensating for uncertainties, errors, and lossesassociated with the assignment process.

Such analysis of multiplex MS/MS spectra may make use of existingalgorithms, such as MASCOT and SEQUEST to subsequently identify each ofthe precursors. A preprocessing step would partition product ions from amultiplex spectrum into multiple virtual MS/MS spectra, each of whichwould contain product ions from only a single precursor. Formation ofvirtual MS/MS spectra according to the invention thus represents“synthetic isolation” of precursors.

A previously described MS/MS demultiplexing method (PCT InternationalPatent Application Publication WO 2008/003684 A1; inventor, Scigocki)has described the use of “correlation laws” to map pairs, triplets, orarbitrarily large subsets of product ions to a precursor ion. Acorrelation law essentially states that the masses of the product ions(formed by multiplying each mass-to-charge ratio by an integerrepresenting the unknown charge of the ion) sum to the mass of theprecursor ion (also formed by multiplying the mass-to-charge ratio bysome integer). However, the observed mass-to-charge ratios containmeasurement errors so that a “proximity criterion” is necessary to allowfor small deviations from the ideal correlation law. In general, becausethe charges for the precursors and products are unknown, there could bea large number of correlation laws (planes passing through the spaceformed by combinations of product mass-to-charge ratios). It isplausible that some of the correlation laws may pass within thetolerance of the observed mass-to-charge ratios of some product ionssimply by random chance leading to false assignments of product ions toprecursors.

From the foregoing discussion, there is a need in the art for improvedmethods and apparatus for obtaining and resolving multiplexed tandemmass spectra. The present invention addresses such a need.

DISCLOSURE OF INVENTION

According to first aspect of the invention, there is provided a firstmethod for obtaining and interpreting multiplex product ion spectra. Thefirst method assumes high mass accuracy spectra of 1) intact precursorions and 2) the product ions that result from simultaneously fragmentingthe precursors. It is also assumed that the masses of both precursorsand products are measured to sufficient accuracy that their elementalcompositions can be determined (or at least reduced to a small number ofpossibilities).

The method computes the probability a given product arose from a givenprecursor for all product-precursor pairs on the basis of aprobabilistic model that assumes no knowledge of the covalent structureof the precursor. In this model, products are generated by uniformlyrandom selection of atoms from the precursor. The resulting distributionof product elemental compositions is multinomial over the various typesof elements occurring in the precursor.

These probabilities are used to assign product ions to precursors, thusgenerating synthetic MS/MS spectra that can be interpreted separately inparallel by existing algorithms. The candidate identifications producedby these algorithms can be combined to form synthetic multiplex production spectra that can be directly matched against the observed multiplexspectrum to determine the most likely set of precursor identifications.

According to a second aspect of the invention, a second method isprovided in which robust detection of pairwise complementary productions uses at least partially-known elemental composition (EC) analysis.In spectra with high mass accuracy and resolving power, the ECs of theproduct and precursor ions can be inferred. When the sum of two production ECs is an exact match to a given precursor ion EC, it is possible toconfidently identify these product ions as complementary and assign themto the corresponding precursor. High mass accuracy and resolving powerenables charge-state and elemental composition determination. Elementalcomposition, in theory, provides an exact match between pairs of productions and precursor ions.

Some embodiments in accordance with the invention comprise methods oftandem mass spectrometry (MS/MS) for use in a mass spectrometer, themethods characterized by the steps of: (a) providing a sample ofprecursor ions comprising a plurality of ion types, each ion typecomprising a respective range of masses; (b) generating a mass spectrumof the precursor ions using the mass spectrometer so as to determine arespective mass value or mass value range for each of the precursor iontypes; (c) estimating an elemental composition for each of the precursorion types based on the mass value or mass value range determined foreach respective ion type; (d) generating a sample of fragment ionscomprising plurality of fragment ion types by fragmenting the pluralityof precursor ion types within the mass spectrometer; (d) generating amass spectrum of the fragment ion types so as to determine a respectivemass value or mass value range for each respective fragment ion type;(e) estimating an elemental composition for each of the fragment iontypes based on the mass value or mass value range determined for eachrespective fragment ion type; and (f) calculating a set of probabilityvalues for each precursor ion type, each probability value representinga probability that a respective fragment ion type or a respective pairof fragment ion types was derived from the precursor ion type. Someembodiments may include a step (g) of generating a synthetic MS/MSspectrum for each respective precursor ion type based on the calculatedprobability values. Some embodiments may further include an additionalstep (h) of providing at least one of the synthetic MS/MS spectra asinput to a peptide identification software product, such as MASCOT orSEQUEST, so as to identify a peptide in a sample from which the sampleof precursor ions is derived.

In some embodiments in accordance with the invention, the step (d) ofgenerating a sample of fragment ions comprising plurality of fragmention types may comprise the steps of: (d1) selecting a subset of theprecursor ion types, the subset comprising a group of precursor iontypes of interest; (d2) isolating a precursor ion type of interest in amass analyzer of the mass spectrometer; (d3) transferring the isolatedprecursor ion type of interest to a collision cell or a reaction cell ofthe mass spectrometer; (d4) repeating steps (d2) and (d3) for eachremaining precursor ion type of interest so as to provide a mixture ofprecursor ion types of interest; and (d5) generating the sample offragment ions by simultaneously fragmenting the precursor ions ofinterest in the collision cell or reaction cell. Alternatively, thefragment ions may be generated by fragmenting the plurality of precursorions simultaneously, possibly in a collision cell or reaction cell.

In some embodiments in accordance with the invention, the step (f) ofcalculating a set of probability values for each precursor may comprisethe steps of: (f1) estimating a variance of the mass of each precursorion type and each fragment ion type; (f2) estimating a variance of amass difference for each possible triplet of ion types, the tripletconsisting of one precursor ion type and two fragment ion types; and(f3) retaining, for consideration, only those triplets of ion types forwhich the mass difference is equal to zero within a certain multiple ofthe respective variance of the mass difference. The following set ofsteps may also be included: (f4) estimating respective elementalcompositions for the precursor ion type and each fragment ion type ofeach retained triplet; (f5) estimating a probability of the correctnessof each respective estimated elemental composition estimated in step(f4); and (f6) calculating a probability that the two fragment ion typeswere formed by fragmentation of the precursor ion type of each retainedtriplet, based on the estimated probabilities of the correctnessestimated elemental compositions.

The mass spectrometer may comprise an ion cyclotron resonance massspectrometers or an Orbitrap mass spectrometer and may provide a massaccuracy of 1 ppm or better. It may comprise a single mass analyzer or,alternatively, a first mass analyzer and a second mass analyzercomprising higher accuracy than the second mass analyzer. In the lattercase, an ion storage device may be provided between the first and secondmass analyzers.

BRIEF DESCRIPTION OF DRAWINGS

The above noted and various other aspects of the present invention willbecome apparent from the following description which is given by way ofexample only and with reference to the accompanying drawings, not drawnto scale, in which:

FIG. 1A is a schematic illustration of a first example of a generalizedtandem mass spectrometer system on which the invention according to someof its aspects may be practiced;

FIG. 1B is a schematic illustration of a second example of a generalizedmass spectrometer system on which the invention according to some of itsaspects may be practiced;

FIG. 1C is a schematic illustration of a third example of a generalizedmass spectrometer system on which the invention according to some of itsaspects may be practiced;

FIG. 2A is a schematic illustration of a particular mass spectrometersystem on which the invention according to some of its aspects may bepracticed, the system including an Orbitrap mass analyzer;

FIG. 2B is a schematic illustration of another particular massspectrometer system on which the invention according to some of itsaspects may be practiced, the system including an Orbitrap massanalyzer;

FIG. 3 is a flow chart of a first method for matching mass spectrometryprecursor and product ions in accordance with the present invention;

FIG. 4A is a flow chart illustrating steps comprising preprocessing ofMS and MS/MS spectra in accordance with a second method for matchingmass spectrometry precursor and product ions in accordance with thepresent invention;

FIG. 4B is a flow chart illustrating steps comprising screening MS andMS/MS spectra for complementary product ions in accordance with thesecond method for matching mass spectrometry precursor and product ionsin accordance with the present invention;

FIG. 4C is a flow chart illustrating steps comprising assessment ofcomplementary product ion pair candidates by elemental compositionanalysis in accordance with the second method for matching massspectrometry precursor and product ions in accordance with the presentinvention.

MODES FOR CARRYING OUT THE INVENTION

The following description is presented to enable any person skilled inthe art to make and use the invention, and is provided in the context ofa particular application and its requirements. Various modifications tothe described embodiments will be readily apparent to those skilled inthe art and the generic principles herein may be applied to otherembodiments. Thus, the present invention is not intended to be limitedto the embodiments and examples shown but is to be accorded the widestpossible scope in accordance with the features and principles shown anddescribed. The particular features and advantages of the invention willbecome more apparent with reference to the appended FIGS. 1-4 taken inconjunction with the following description.

A first example of a generalized tandem mass spectrometer system 100 onwhich the invention according to some of its aspects may be practiced isshown in FIG. 1A. Analyte material 105 is provided to a pulsed orcontinuous ion source 110 so as to generate ions 115. The ions areadmitted to a first mass analyzer (MS-1) 120 that has mass analysis andmass selection functionality and in which, optionally, fragmentation maybe performed. For instance, the first mass analyzer MS-1 may comprise anion trap. Alternatively, a separate reaction cell (not shown in FIG. 1A)may be used to perform fragmentation. Ion source 110 could be a MALDIsource, an electrospray source or any other type of ion source. Inaddition, multiple ion sources may be used. Also, the mass analyzer MS-1120 may be preceded by any number of other stages of mass analysis,and/or ion manipulation.

It is to be noted that, in the system of FIG. 1A as well as in systemsillustrated in subsequent drawings, ions are transferred from onecomponent to the next via ion optics (e.g., RF multipoles) which, inmost cases, are not specifically illustrated. Moreover, the drawings donot show the electrodes of the various parts-that are used to guideand/or trap ions within those parts.

All embodiments of the invention may be operated with an automatic gaincontrol (AGC) detector 130 (see FIG. 1A, for instance) to trap anappropriate number of ions. Any of the known AGC methods may be used todetermine the optimum ionization time for fills of the downstreamintermediate ion storage 140 or the accurate mass analyzer MS-2 170.Accordingly, a proportion of ions exiting MS-1 may be diverted alongpath 125 c to AGC detector 130. Otherwise, ions are transferred fromMS-1 along path 125 a to the intermediate ion storage 155.

In this application, AGC is interpreted in a most general way as amethod of determining an optimum fill time based on sampling a set ofions. Therefore, it includes not only methods based on information froma pre-scan or previous scan, but includes other methods of measuringnumbers of ions such as a current sensing grid that intercepts(preferably uniformly) an ion beam; sensing induced currents; sensingscattered ions, for example on apertures; sensing secondary electrons;and using a previous analytical scan taken by the first mass analyzer120. Ions produced using the optimum ionization time may be fragmentedin either the first mass analyzer 120 or a separate reaction cell, forexample, by collision-induced dissociation.

Selected ions are transferred from the first mass analyzer 120 alongpath 125 a into the intermediate ion storage device 140 where they arecaptured and trapped. The intermediate ion storage device 140 maycomprise, for instance, an ion trap device. Ions released from theintermediate ion storage device 140 are transferred along path 155 to anaccurate mass analyzer (MS-2) 170. The accurate mass analyzer mayreceive, for analysis, either unfragmented precursor ions, a set of ionsformed by fragmentation of a single selected precursor ion, or a mixtureof a plurality of sets of ions, each such set formed by fragmentation ofa respective precursor ion. The accurate mass analyzer has sufficientlyhigh m/z resolution to resolve all species in such mixed ionpopulations. Examples of suitable accurate mass analyzers are ioncyclotron resonance mass spectrometers and Orbitrap (a type ofelectrostatic trap) mass spectrometers.

Continuing with the discussion of FIG. 1A, a controller 160, which maycomprise a general purpose computer or, perhaps, a specializedelectronic logic device, is electronically coupled to all othercomponents along electronic control lines 175. The electronic controllines 175 may send control signals from the controller 160 to the massspectrometers, intermediate ion storage device, ion source, the variousion optics, etc. in order to control the coordinated operation of thesecomponents. For instance, the controller may send signals to setpotentials on the electrodes of the various parts at the variousappropriate times. The electronic control lines 175 may also transmitsignals from one or more of the components of the system 100 back to thecontroller 160. For instance, the controller 160 may receive signalsfrom the AGC detector 130 and from the accurate mass analyzer 170, suchsignals relating to number of ions detected.

A second example of a generalized mass spectrometer system 200 on whichthe invention according to some of its aspects may be practiced is shownin FIG. 1B. The system 200 shown in FIG. 1B comprises all of thecomponents as described with reference to the mass spectrometer system100 (FIG. 1A), with similar reference numbers thus being used in the twodrawings. With reference to FIG. 1B, however, it is to be noted that,although most components of the mass spectrometer system 200 arepositioned on the longitudinal “axis” 107 (shown as a dot-dash line),the accurate mass analyzer MS-2 is positioned off of this axis. Further,a reaction cell for fragmentation of ions is disposed along axis 107 atthe side of the intermediate ion storage device opposite to MS-1.Although the curve 107 is shown as a straight line and referred to as an“axis”, it should be noted that, in practice, at least a portion of thiscurve may not, in fact, be linear.

The system 200 shown in FIG. 1B (and also the system 300 shown in FIG.1C) provides for two types of ion pathways between the first massanalyzer MS-1 and the accurate mass analyzer MS-2, corresponding to tworespective modes of operation. In a first mode of operation, selectedions are delivered along pathway 125 a from MS-1 to the intermediate ionstorage device 140 where they are trapped. Once a suitable time delayhas passed, the controller 160 transports the ions to the reaction cell150. In a second, alternative, mode of operation, the intermediate ionstorage device 140 is used merely as an ion guide (“transmission mode”)such that ions are transferred along pathway 125 b (which may, in factbe coincident with path 125 a but which is shown offset from thatpathway, for clarity) from MS-1 to the reaction cell 150. Theintermediate ion storage device 140 may be filled with gas, therebyreducing the energy of the ions through collisional cooling as they passthrough the intermediate ion storage device and enter the reaction cell150.

Precursor ions may be fragmented in the reaction cell. Ion fragmentationmay be effected by any suitable fragmentation technique, such ascollision-induced dissociation (CID), electron transfer dissociation(ETD), electron capture dissociation (ECD) or infrared multiphotondissociation (IRMPD). The resulting fragment ions (if any) or precursorions (if any) are then transferred, in the opposite direction, backalong path 125 b from the reaction cell to the intermediate ion storagedevice 140. After storage in the intermediate ion storage device 140 foran appropriate time, these fragment ions are transferred to the accuratemass analyzer 170 for analysis along pathway 155. Multiple fills of theaccurate mass analyzer 170 may be formed using different respectiveprocessing techniques (for instance, high energy versus low energyfragmentation) in the reaction cell 150. This flexibility provides thecapability of performing both precursor ion as well as fragment ionanalyses using the accurate mass analyzer.

Automatic gain control, as facilitated by the AGC detector 130, may beused to control the ion abundance in the intermediate ion storage device140, the reaction cell 150 or the accurate mass analyzer 170. Automaticgain control is described in U.S. Pat. Nos. 5,107,109 and 6,987,261,both of which are incorporated by reference herein in their entirety.

FIG. 1C illustrates a third example of a generalized mass spectrometersystem 300 on which the invention according to some of its aspects maybe practiced. The mass spectrometer system 300 is similar to the system200 illustrated in FIG. 1B, except that the system 300 does not comprisea first mass spectrometer MS-1. Thus, in the system 300, the ion source110 delivers, to either or both of the intermediate ion storage device140 and the reaction cell 150, streams or pulses of ions which are notpre-selected or pre-isolated according to their m/z.

FIG. 2A is a diagram of a particular example of a mass spectrometersystem of the type earlier shown in FIG. 1B. In the mass spectrometer400 shown in FIG. 2A, the intermediate ion storage device comprises acurved quadrupolar linear ion trap (shown as reference number 140-c)bounded by gates 142 at respective ends. The curvature of theintermediate ion storage device 140-c is used such that, when the ionsare ejected off axis, the ions are radially convergent. The ions areejected off-axis in the direction of the entrance 172 to an Orbitrapmass analyzer 170-o, which serves as the accurate mass analyzer in thisexample. The ions are ejected from the curved trap 140-c through anaperture 148 provided in an electrode 146 of the curved trap 140-c andthrough further ion optics 157 that assist in focusing the emergent ionbeam. It will be noted that the curved configuration of the intermediateion storage device (i.e., the curved quadrupolar linear ion trap 140-cin this particular instance) also assists in focusing the ions. Thecurved linear ion trap 140-c is inherently useful as it allows rapidejection of pulses of ions to the mass analyzer 170-o with little, ifany, further shaping required.

In operation, ions are generated in the ion source 110 and transportedthrough ion optics so as to be accumulated temporarily in MS-1 120according to e.g. US20030183759 or U.S. Pat. No. 6,177,668. MS-1 120 maycontain an inert gas (i.e., 1 mTorr of helium) such that the ions losesome of their kinetic energy in collisions with the gas molecules.

Either after a fixed time delay (chosen to allow sufficient ions toaccumulate in MS-1 120) or after sufficient ions have been detected inMS-1 120 (possibly through detection with AGC detector 130), ions areejected from MS-1 120 so as to travel into the intermediate ion storagedevice 140-c. As discussed previously, ions may pass through theintermediate ion storage device 140-c into the reaction cell 150 wherethey are processed before being returned back to the intermediate ionstorage device 140-c.

FIG. 2B is a diagram of a particular example of a mass spectrometersystem of the type earlier shown in FIG. 1C. The system 500 shown inFIG. 2B comprises the components shown in FIG. 2A except for the firstmass analyzer MS-1. Thus, ions of a range of m/z may be passed throughto the intermediate ion storage device 140-c, to the reaction cell 150and to the accurate mass analyzer 170-o.

In the following discussion, inventive algorithms are described whichprovide the enabling technology for MS/MS multiplexing: matching productions observed in a multiplex MS/MS spectrum to precursor ions observedin an MS spectrum. As used in this specification, an “ion type” includesall ions having the same charge state and identical numbers of atoms ofeach element—for instance, the same number of carbon atoms, the samenumber of nitrogen atoms, etc. Frequently, each ion type may comprise arespective range of masses because of the distribution of differentisotopes of the various atoms within the atom. Occasionally, however, anion type may consist of a single, discrete mass. For instance, massspectra may not exhibit isotope peaks if a single mass-to-charge ratiowas previously selected and isolated. A monoisotopic peak representsonly the principal isotopes of the atoms of which the ion is composed.

The fundamental challenge of the “all ions” workflow is theinterpretation of the multiplex fragmentation spectrum. The problem isanalogous to spilling the pieces from a stack of puzzle boxes into apile and trying to assemble all the puzzles at the same time. Oneapproach to the problem is use clues about the puzzle pieces to placeeach piece back into its box. If this could be accomplished, then theproblem can be solved by repeatedly assembling a single puzzle from itspieces. Likewise, if there were a mechanism for mapping each product ionto its precursor ion then existing methods for MS/MS analysis of productions from isolated precursors could be used repeatedly to identify eachprecursor.

EXAMPLE 1 Elemental Compositions Known

Continuing the puzzle analogy, the puzzle pieces may contain clues thatallow them to be grouped correctly into families, including the textureor color on the backs of the pieces, the material, or the distributionof colors or sizes. At first glance, ions would not seem to provide suchclues. However, the elemental compositions of ions would containinformation about their precursor ion of origin. For example, a production containing a sulfur atom cannot arise from a precursor withoutsulfur. Similarly, a product ion containing six nitrogen atoms cannotarise from a precursor atom containing five or fewer nitrogen atoms.These are examples that place absolute constraints in the mapping ofcertain products to precursors. In general, the precursor must containat least as many atoms of each elemental type as appears in its putativeproducts. In cases where this criterion is not satisfied, theprobability that the product originated from that precursor is exactlyzero.

The analysis can be further generalized to include statements orrelative, rather than absolute, probability. For example, a product thatis almost identical to its precursor (e.g., differs by a single methylgroup or a single amino acid residue) is highly likely to haveoriginated from the precursor even though it is possible that it mayhave originated from a much larger molecule.

Consider two precursor molecules A: C₁₀H₁₄O₆ and B: C₃₀H₆₀N₆O₄ and aproduct X: C₈H₁₀O₄. Although it is possible that product X came fromprecursor B, it is considerably more likely to have come from precursorA, based upon their elemental compositions and the laws of probability.If no structural information is available about A and B, thedistribution of product elemental compositions could be modeled byselecting atoms from the precursors at random. In that case, elements inthe product would tend to occur in similar proportions as in theprecursor. In the present situation, production of product X by randomlyselecting 22 of the 100 atoms from B would be unlikely to result in acollection that contains none of its six nitrogens and all of its fouroxygens. In contrast, the selection of 8 of 10 carbons, 10 of 14nitrogens, and 4 of 6 carbons (as would be required if X was a productof A) is a much more likely outcome.

The inventive method formalizes the reasoning described above toevaluate the probability that a given product would have arisen fromeach of N possible precursors given only the elemental composition ofthe product and the precursors. First, consider the distribution ofproducts that would arise from a given precursor. Assume that theproduct and precursor are identified only by their elementalcompositions. That is, no structural information is available abouteither the precursor or the product. In other words, all structures areequally likely.

In this case, the distribution of products is mathematically equivalentto the outcomes of drawing colored balls from an urn withoutreplacement. The balls placed in the urn (before drawing any out)represent atoms in the precursor. The colors of the balls placed in theurn are chosen to represent the different elemental types of atoms thatoccur in the precursor; the number of balls of a given color is chosento match the number of atoms of the corresponding type occurring in theprecursor. Balls drawn at random out of the urn without replacementrepresent the atoms that would occur in a randomly generated product.For example, the distribution of products containing exactly N atomscould be generated by drawing N balls from the urn without replacementand repeated such a trial of N selections a large number of times.Fortunately, it is possible, and straightforward, to calculate thedistribution of outcomes in the limit of an infinite number of trialsfor an arbitrary value of N (the number of atoms in the product) and anarbitrary precursor elemental composition.

For example, consider a precursor “α” made up of atoms of K differenttypes: α₁ atoms of type 1, α₂ atoms of type 2, and in general, α_(k)atoms of type k, where k is an integer between 1 and K. Assuming that itis possible to specify the types of atoms 1 to K, then the elementalcomposition of α can be represented by the K-component vector a=α₁, α₂,. . . , α_(K)). Each α_(k) must be a positive integer.

Likewise, consider a potential product “X” made up of the same K typesof atoms as the precursor. The elemental composition of X may berepresented by the vector x=(x₁, x₂, . . . , x_(K)). For X to be acandidate product of α, each x_(k) must be a non-negative integer withthe constraint that x_(k)<=α_(k). For notational shorthand, let A be thesum of the α_(k)'s (Equation 1) and let X be the sum of the x_(k)'s(Equation 2). Then, A and X denote the number of atoms in X and αrespectively.

$\begin{matrix}{A = {\sum\limits_{k = 1}^{K}\; a_{k}}} & (1) \\{X = {\sum\limits_{k = 1}^{K}\; x_{k}}} & (2)\end{matrix}$The probability of generating product X with elemental composition xfrom precursor α is given by Equation 3: An additional constraint isthat the product has X atoms.

$\begin{matrix}{{P\left( {\left. x \middle| a \right.,X} \right)} = \frac{\prod\limits_{k = 1}^{K}\;\begin{pmatrix}a_{k} \\x_{k}\end{pmatrix}}{\begin{pmatrix}A \\X\end{pmatrix}}} & (3)\end{matrix}$The denominator of the above equation denotes the number of ways to drawX atoms from A atoms. Each factor in the numerator gives the number ofways to draw X_(k) atoms of type k from α_(k) atoms of type k. When A isdivisible by X, it can be shown that the most likely product is (X/A)a.That is, the most likely product is one that has atoms occurring in thesame proportion as the precursor. In general, when A is not divisible byX, the most likely product(s) are vectors with integer components thatare “closest” to the vector (X/A)a. The equation above can be thought ofas a mathematical statement of the intuitive notion that products havecompositions that are similar to their precursor.

Equation 3 above states the distribution of products containing exactlyX atoms. Equation 4 below gives the distribution of products ofarbitrary size. The product on the right-hand side reflects thatgenerating a product can be modeled abstractly as two sequentialindependent processes: selecting a product size X and then selecting aproduct elemental composition x, composed of X atoms. The first-termp(X/A) is the probability that a precursor of A atoms would produce aproduct of X atoms. The product size is assumed to depend only on thesize of the precursor, and not its elemental composition.P(x|a)=P(X|A)P(x|a,X)  (4)Without knowledge of the precursor structure, the most reasonableassumption about the product size distribution is uniformity. That is,the probability of X is 1/A for all X between 1 and A, and zerootherwise (Equation 5).

$\begin{matrix}{{P\left( X \middle| A \right)} = \left\{ \begin{matrix}{1/A} & {x \in \left\{ {1,\ldots\mspace{14mu},A} \right\}} \\0 & {otherwise}\end{matrix} \right.} & (5)\end{matrix}$For example, a uniform distribution of product sizes would be generatedby selecting a bond uniformly at random from the linear precursorstructure at random and breaking it. Likewise, a uniform distribution ofproduct sizes would result from first selecting a randomly generatedprecursor structure of a given elemental composition and then breaking arandomly selected bond. Equation 6 results from inserting into Equation4 the uniform factor given in Equation 5.

$\begin{matrix}{{P\left( x \middle| a \right)} = {\frac{1}{A}{P\left( {\left. x \middle| a \right.,X} \right)}}} & (6)\end{matrix}$

More-realistic distributions of product sizes could be used instead ofthe uniform distribution to take into account various effects, eitherobserved or theoretical, that impose a bias upon observed product sizes.For example, a smaller molecule would be less likely to contain acharge-carrying site than a larger molecule. However, a larger ion wouldbe more likely to contain an unstable bond that would eliminate theintact species before it could be observed. Without a more detailedanalysis, it is not clear which of these effects would be moresignificant.

Another refinement of the model is a consideration of charge mobility.In the case of an immobile charge, the ion's charge state would beconsidered as component k+1 of the vector. Thus, the charge on the ionwould tend to partition in the same way as the atoms, so that a productthat is half the size of the precursor would be most likely to have halfits charge. However, if the charge is absolutely mobile, spending anequal amount of time associated with any atom, then all products couldbe observed. Smaller products would be seen at proportionately lowerabundance than larger products since the probability that the charge wasresiding in a given region of the molecule at the instant offragmentation would vary in proportion to the product size.

Equation 6 above provides the distribution of products that a givenprecursor would produce. It is an intermediate step in computing theprobability that an observed product originated from a particularprecursor (i.e., the desired quantity). The latter quantity may bederived in terms the former expression (derived above) by using Bayes'Theorem. The result is shown in Equation 7.

$\begin{matrix}{{P\left( a \middle| x \right)} = \frac{{P(a)}{P\left( x \middle| a \right)}}{\sum\limits_{a^{\prime}}\;{{P\left( a^{\prime} \right)}{P\left( x \middle| a^{\prime} \right)}}}} & (7)\end{matrix}$In the above equation, vectors a and x denote the elemental compositionof one of the observed precursors and observed products respectively.The denominator is a normalizing factor that is the sum over allobserved precursor elemental compositions. These precursors are indexedby the variable a′. The expression P(a|x) is evaluated using theequation derived above for each pair (x, a) formed by selecting one ofthe observed products and one of the observed precursors.

An important special case is that all precursors are equally likely. Inthis case, the value of P(a) is equal for all candidates and theexpression for the probability is given by Equation 8.

$\begin{matrix}{{P\left( a \middle| x \right)} = \frac{P\left( x \middle| a \right)}{\sum\limits_{a^{\prime}}\;{P\left( x \middle| a^{\prime} \right)}}} & (8)\end{matrix}$The above equation assigns probabilities to the candidate precursorsthat sum to one. However, the equation does not take into account thepossibility that the product comes from none of candidates or from morethan one of them. These considerations are relatively minor effects thatdo not significantly diminish the utility of the calculated probabilityestimates in most cases.

Note that uncertainties in determining the elemental composition ofeither the precursors or the product ions can also be accommodated bycomputing the probability-weighted sum of either Equation 7 or 8, wherethe sum is taken over the candidate elemental compositions and eachweight is the probability associated with a particular candidate.

FIG. 3 is a flow chart of a first method, method 600, in accordance withthe present invention and the above discussion. The method 600 providesan implementation of the calculations discussed above for matching massspectrometry precursor and product ions. In the initial step, step 302,a sample of interest is ionized and these ions and fragments thereof areanalyzed by tandem mass spectrometry. This analysis yields the masses ofa set of M sample-derived ions (precursor ions) and a set of N product(fragment) ions produced by decomposition of the precursor ions. Fromthese experimentally obtained masses, the elemental compositions of eachprecursor ion and each product ion may, in some instances, be assigned.This yields a set of M precursor elemental compositions and N productelemental compositions in addition to the M precursor masses and Nproduct masses determined by the mass analysis.

The lists of precursor and product ionic masses and elementalcompositions obtained in step 302 of the method 600 (FIG. 3) areutilized in calculation steps 304-318. The step 304 is an initiationstep for a first loop. In step 304, each precursor ion, α_(i), ispresented for consideration in sequence, such that each iteration of thesubsequent steps in the loop, steps 306-320 produces a set ofassignments of the product ions most likely to have been produced byfragmentation of α_(i).

In step 306, the value of A_(i) (the appropriate value of A for theprecursor ion α_(i)) is calculated from Eq. 1. Subsequently, step 308 isan initiation step for a second loop (an inner loop) that is nestedwithin the first loop. In step 310, each product ion, X_(j), ispresented for consideration in sequence, such that each iteration of thesubsequent steps 312-316 yields a numerical probability that the production under consideration, X_(j), was produced by fragmentation of α_(i).In step 310, the value of X_(j) (the appropriate value of X for theprecursor ion X_(j)) is calculated from Eq. 2. Subsequent steps 312, 314and 316 respectively yield calculations of P(x_(j)|a_(i),X_(j)) (Eq. 3),P(x_(j)|a_(i)) (Eqs. 4-6) and P(a_(i)|x_(j)) (Eqs. 7-8), where thevectors a_(i) and x_(j) are the coefficient vectors, as previouslydefined, for the particular precursor ion, α_(i), and particular production, X_(j), respectively.

The calculated probabilities P(a_(i)|x_(j)) can be used todeterministically (i.e., maximum likelihood) or randomly (i.e., MonteCarlo sampling) assign products to precursors for downstream analysis.FIG. 3 provides one example of such an assignment. In this example,after steps 308-316, have been completed then, if P(a_(i)|x_(j)) isfound, in step 318, to be above a certain possibly pre-defined thresholdvalue, T, the product ion under consideration is assigned as having beenproduced from precursor ion α_(i). The set of such assignments yield asynthetic MS/MS spectrum for the precursor ion α_(i) in step 320.Subsequently, the method 600 loops back to step 304 in which anotherprecursor ion is presented for consideration and the steps 306-320 areexecuted once again using this new precursor ion.

The set of products assigned to a given precursor can be thought of assynthetic MS/MS spectrum. The synthetic spectrum can be presented to anMS/MS identification program like Mascot or SEQUEST as if it were anobserved spectrum. Analysis of the best hits can be used to update theprobability estimates and iteratively redistribute the products amongthe precursors.

EXAMPLE 2 Pairwise Correlation Using at Least Partially Known ElementalCompositions

A feature of the following algorithm is the robust detection of pairwisecomplementary product ions using at least partially-known elementalcomposition (EC) analysis. In many cases, a precursor ion fragments intotwo stable product ions that are both detectable in an MS/MS spectrum.In spectra with high mass accuracy and resolving power, the ECs of theproduct and precursor ions can be inferred. When the sum of two production ECs is an exact match to a given precursor ion EC, it is possible toconfidently identify these product ions as complementary and assign themto the corresponding precursor.

First, a sample comprising a mixture of parent ions is analyzed bytandem mass spectrometry using a mass spectrometer system such as isillustrated in FIGS. 1-2. The system is used so as to detect bothprecursor and product ions, possibly by performing separate precursorion scans (to generate MS spectra) and product ion scans (to generatetandem or MS/MS spectra) as described above. In such a case, separateprecursor ion and product ion mass spectra are obtained, the precursorspectrum containing peaks relating to a plurality of parent ions and theproduct spectrum containing “multiplexed” peaks relating tofragmentation products of all of or many of the precursors.Alternatively, the mass spectra may be obtained by performing partialfragmentation such that both precursor and product ion peaks are presentin a single mass spectrum. In this case, precursor ions are identifiedby their much greater masses relative to the fragments.

Subsequently, the mass spectra are analyzed by the algorithm describedbelow, comprising three phases: a first preprocessing phase (FIG. 4A) inwhich the mass, charge and variance of the mass is estimated for eachrespective precursor ion peak and product ion peak in the mass spectra;an accurate mass screen (FIG. 4B) for candidate pairs of complementaryions and a final phase (FIG. 4C) comprising a more rigorous test ofthese candidates using elemental composition analysis. The screeningstep is present to improve the time performance of the algorithm.

The preprocessing phase, specifically method 700 illustrated in FIG. 4A,comprises two separate stages—a first stage comprising steps 402 through410 relating to precursor (parent) ions and a second stage comprisingsteps 412 through 420 relating to product ions. As may be observed fromFIG. 4A, the steps are similar between the two stages. The two stagescould be performed sequentially or simultaneously.

The steps 402 and 412, respectively relating to identification andextraction of precursor and product ion peaks from a single massspectrum or from separate precursor and product mass spectra, arestandard operations and have already been discussed. The next steps(step 404 and step 414) comprise identifying the monoisotopic peaks. Inthis regard, it is also assumed that the spectral peak corresponding tothe monoisotopic species can be unambiguously determined. Then, for eachmonoisotopic ion (of mass M_(k) for each of K precursor ions in step 406or of mass m_(i) for each of I product ions in step 416), determinationsare made, in sequence, of M_(k)/z_(k) (or m_(i)/z_(i)), of z_(k) (orz_(i)) and finally, of M_(k) and σ_(k) (or m_(i) and σ_(i)). Thesedeterminations are made in steps 408 through 410 for precursor ions andin steps 418 through 420 for product ions. The values of M_(k)/z_(k) andm_(i)/z_(i) (steps 408 and 418) may be derived directly from the massspectra, using well-known calibration methods. The next analytic stepsin the algorithm (steps 409 and 419) are the determination of thecharge-state of each monoisotopic ion. For purposes of thisdetermination, it is assumed that sufficient resolving power exists toresolve isotopic species that differ by one neutron. The difference inmass-to-charge ratio of such species is equal to the inverse of thecharge. Therefore, the charge is determined by the inverse of the m/zspacing between adjacent peaks that are identified as isotopicallyrelated. Given the charge of the ion, the monoisotopic ion's measuredmass-to-charge ratio can then be easily converted into an estimate ofthe mass of the neutral species (steps 410 and 420). In addition toestimates of neutral monoisotopic masses, it is also assumed that theuncertainties of the mass estimates are known or can be estimated.

Given the list of mass estimates and their uncertainties obtained asdescribed above, it is possible to generate a list of candidate triplets(precursor, product₁, product₂) that could be related by thefragmentation reactionprecursor→product₁+product₂The magnitude of the estimated difference between the precursor mass andthe sum of the two product masses should be similar to the uncertaintiesin the mass measurements. In particular, if (M_(k),σ_(k) ²),(m_(i),σ_(i) ²), and (m_(j),σ_(j) ²) denote the estimated mass and itsvariance for the precursor and two products respectively, then thedifference M_(k)−(m_(i)+m_(j)) would have variance σ_(k) ²+σ_(i) ²+σ_(j)². If the mass errors are normally distributed with zero mean, than itis statistically expected that more than 99% of related masses havedifferences less than three sigmas from the mean, i.e. 3 (σ_(k) ²+σ_(i)²+_(j) ²)^(1/2). Therefore, a threshold on the mass difference is usedas a criterion for selecting candidates.

With the above background, it is possible to efficiently detectcandidates from the list of precursor and product masses with a searchalgorithm (illustrated in FIG. 4B as method 800) that has computationalcomplexity KN log N, where K and N are the number of precursor andproduct ions respectively, as described below. The steps 502 through 511of method 800, which comprise an outer loop are performed for eachprecursor ion (e.g., with mass M_(k)). The steps 504 through 511, whichcomprise an inner nested loop, are performed for each product ion (e.g.,with mass m_(i)). Thus, for each precursor and product ion, a search isconducted for other product ions (steps 506 through 511, comprising aninnermost nested loop) whose mass is (approximately) equal to the massdifference M_(k)−m_(i) within statistical limits. The search may beperformed as a binary search on a list of product ions sorted by mass.The search (requiring at most log N steps) returns the two product ionswhose masses bracket the required mass difference of the complementaryfragment. If either of these masses differs from the target by less thanthe threshold (either step 508 or step 510), the triplet is retained asa candidate (steps 509 and 511).

It is reasonably expected that the number of product ions should belarger than the number of precursor ions (i.e., N>>K), so the methoddescribed above would be faster than forming the pairwise sums of allproduct ions and then searching against the sorted precursor ion lists.This alternative suboptimal method would have complexity N² log K (>>KNlog N).

In the next phase (FIG. 4C), the candidate triplets generated by themethod 800 (FIG. 4B) are more rigorously screened by elementalcomposition determination. For example, suppose the elementalcompositions of the precursor and two product ions are denoted by E, e₁,and e₂ respectively. The quantities E, e₁, and e₂ are vectors whosecomponents are integer values specifying the number of atoms of variouselemental types present in the respective neutral species. If theproduct ions are complementary fragments resulting from the precursorion, their elemental compositions must sum (exactly) to the precursorelemental composition, i.e., E=e₁+e₂. Equality of the vector sumrequires equality of each component sum.

The isotope envelope is then used to assign probability to candidateelemental compositions for the precursors and products. The probabilitythat the three indicated ions form a set related by a fragmentationreaction is given by Eq. 9

$\begin{matrix}{P = {\sum\limits_{E = {e_{2} + e_{2}}}\;{{p(E)}{p\left( e_{1} \right)}{p\left( e_{2} \right)}}}} & \left( {{Eq}.\mspace{14mu} 9} \right)\end{matrix}$Each term in the probability sum is the product of three probabilityfactors, each indicating the probability that a given elementalcomposition is correct. The terms in the sum reflect different possiblecombinations of elemental compositions that sum together as required bythe fragmentation reaction.

Elemental composition determination is not a routine application in massspectrometry. However, it should be noted that the potential number ofelemental compositions increases rapidly with mass. So, in most cases,elemental composition determination is much more definitive for productions than for precursor ions. The elemental compositions of some productions cannot be determined with high confidence. Even though it may notbe possible to exactly identify the elemental composition of a precursorion, the observed isotope envelope often provides sufficient informationto count heteroatoms, e.g., sulfur, or to count carbon atoms within10-20% accuracy. In some cases, there is a priori information aboutpossible elemental composition or molecular structure, e.g., proteomicor metabolic biotransformation databases. In combination, theseconstraints on product and precursor ions provide confident verificationof a complementary relationship between them.

The method 900 illustrated in FIG. 4C formalizes these procedureoutlined above. For each of the candidate triplets generated by themethod 800 (FIG. 4B), there may exist several candidate elementalcompositions (EC's), including candidate EC's for the candidateprecursor and for each of the candidate products. The steps 602 through616 form an outermost loop in which each candidate triplet (M_(k),m_(i), m_(j)) is evaluated in turn. Each of steps 606, 608 and 610initiates a progressively nested inner loop in which candidatecompositions are considered for the candidate precursor ion and for eachof the candidate product ions, respectively. The result of thisevaluation is, for each candidate triplet, the product P as given byequation Eq. 9. Step 612 calculates each term in the sum of Eq. 9 andadds it to the total sum calculated to that point. In step 614, if theprobability P exceeds a certain pre-defined threshold T, then, in step616, the product ions of the candidate triplet are identified as arisingfrom the precursor ion.

Correct demultiplexing of a subset of the product ions, assigning themto their precursor ions, generates a collection of virtual MS/MSspectra, analogous to spectra that would be formed by the isolatedproduct ions of each fragmented precursor. These virtual spectra areexpected to contain fewer product ions than an actual MS/MS spectrumformed from an isolated precursor. Some product ions result from a“neutral loss” mechanism in which the complementary fragment isnon-ionizable, and thus not detected by a mass spectrometer. Inaddition, other complementary fragments may be unstable, and thus notpresent at detectable levels. In other cases, the complementary fragmentmay be too small to be detected, i.e., below the lower limit of thespectrum's mass range. In each case where the complementary fragment isnot detected, its partner fragment that appears in the actual isolatedMS/MS spectrum is lost in the virtual demultiplexed MS/MS spectrum.

By assigning pairs of complementary product ions to precursors, themultiplex MS/MS spectrum is demultiplexed to form “virtual” MS/MSspectra, each corresponding to an MS/MS spectrum from an isolatedprecursor. Each virtual MS/MS spectrum can be submitted to standardalgorithms, such as MASCOT and SEQUEST, which identify precursors fromMS/MS spectra. Despite the multiple mechanisms of product ion lossdescribed above, there is often enough product ions in demultiplexedspectra to provide confident precursor identification (cite Zubarev.)

EXAMPLE 3 Experimental Implementation

One proposed experiment of this type performs multiple injections ofdistinct precursor ions, each individually isolated, to create a mixtureof precursor ions that are simultaneously fragmented and analyzed.Another experiment performs coarse isolation (e.g., selecting ionsresiding in a band of tens to hundreds of m/z units) to create a mixtureof precursor ions whose products are analyzed together as before. Athird type of experiment involves the Exactive™ mass spectrometer, astandalone Orbitrap mass analyzer, which does not provide capability forisolation before fragmentation of ions in its HCD collision cell andsubsequent analysis.

The workflow described on the Exactive mass spectrometer provides theability to perform detailed identification and accurate quantificationby alternating two types of scans at high frequency (e.g. 5 Hz). Thefirst scan type is a precursor scan in which ions flow directly from theion source into the analytic cell. The second scan type is an “all ions”fragmentation scan in which all ions (without mass filtering) flow intothe HCD cell (i.e., reaction cell 150) where they are broken intoproducts by collisions with neutral gas molecules. The resultingproducts are then transported into the analytic cell. The analysis ofprecursors at a high scan rate (combining every other scan, i.e., scansof only the first type) allows accurate integration of chromatographicpeak shapes at decreased run times. The analysis of products for allprecursors provides extensive identification coverage.

The discussion herein is intended to serve as a basic description.Although the invention has been described in accordance with the variousembodiments put forth herein, one of ordinary skill in the art willreadily recognize that there could be variations to the embodiments andthose variations would be within the scope of the present invention. Thespecific discussions herein may not explicitly describe all embodimentspossible; many alternatives are implicit. Accordingly, manymodifications may be made by one of ordinary skill in the art withoutdeparting from scope and essence of the invention. Neither thedescription nor the terminology is intended to limit the scope of theinvention. Any patents, patent application publications or otherpublications are hereby explicitly incorporated herein by reference intheir entirety as if set forth fully herein.

1. A method of tandem mass spectrometry (MS/MS) for use in a massspectrometer characterized by the steps: (a) providing a sample ofprecursor ions comprising a plurality of ion types, each ion typecomprising a respective mass or range of masses; (b) generating a massspectrum of the precursor ions using the mass spectrometer so as todetermine a respective mass value or mass value range for each of theprecursor ion types; (c) estimating an elemental composition for each ofthe precursor ion types based on the mass value or mass value rangedetermined for each respective ion type; (d) generating a sample offragment ions comprising plurality of fragment ion types by fragmentingthe plurality of precursor ion types within the mass spectrometer; (e)generating a mass spectrum of the fragment ion types so as to determinea respective mass value or mass value range for each respective fragmention type; (f) estimating an elemental composition for each of thefragment ion types based on the mass value or mass value rangedetermined for each respective fragment ion type; and (g) calculating aset of probability values for each precursor ion type, each probabilityvalue representing a probability that a respective fragment ion type ora respective pair of fragment ion types was derived from the precursorion type.
 2. A method of tandem mass spectrometry as recited in claim 1,further characterized by the step: (h) generating a synthetic MS/MSspectrum for each respective precursor ion type based on the calculatedprobability values.
 3. A method of tandem mass spectrometry as recitedin claim 2, further characterized by the step: (i) providing at leastone of the synthetic MS/MS spectra as input to a peptide identificationsoftware product so as to identify a peptide.
 4. A method of tandem massspectrometry as recited in claim 1, wherein the step (d) of generating asample of fragment ions comprising plurality of fragment ion typescomprises the steps: (d1) selecting a subset of the precursor ion types,the subset comprising a group of precursor ion types of interest; (d2)isolating a precursor ion type of interest in a mass analyzer of themass spectrometer; (d3) transferring the isolated precursor ion type ofinterest to a collision cell or a reaction cell of the massspectrometer; (d4) repeating steps (d2) and (d3) for each remainingprecursor ion type of interest so as to provide a mixture of precursorion types of interest; and (d5) generating the sample of fragment ionsby simultaneously fragmenting the precursor ions of interest in thecollision cell or reaction cell.
 5. A method of tandem mass spectrometryas recited in claim 1, wherein the mass spectrometer provides a massaccuracy of 1 part-per-million or better.
 6. A method of tandem massspectrometry as recited in claim 1, wherein the step (g) of calculatinga set of probability values for each precursor ion comprises the steps:(g1) identifying monoisotopic masses of the mass spectrum of theprecursor ion types and the mass spectrum of the fragment ion types;(g2) estimating a variance of the monoisotopic mass of each precursorion type and each fragment ion type; (g3) estimating a variance of amonoisotopic mass difference for each possible triplet of ion types, thetriplet consisting of one precursor ion type and two fragment ion types;and (g4) retaining, for consideration, only those triplets of ion typesfor which the monoisotopic mass difference is equal to zero within acertain multiple of the respective variance of the mass difference.
 7. Amethod of tandem mass spectrometry as recited in claim 1, wherein thestep (g) of calculating a set of probability values for each precursorion comprises the steps: (g1) selecting certain of the precursor iontypes and certain of the fragment ion types for consideration; (g2)estimating a plurality of elemental compositions for each selectedprecursor ion type and each selected fragment ion type; (g3) estimatinga probability of the correctness of each respective estimated elementalcomposition estimated in step (f2); and (g4) calculating a probabilitythat each pair of the selected fragment ion types was formed byfragmentation of each one of the selected precursor ion types, based onthe estimated probabilities of the correctness estimated elementalcompositions.
 8. A method of tandem mass spectrometry as recited claim6, wherein the step (g) of calculating a set of probability values foreach precursor ion further comprises the steps: (g5) estimatingrespective elemental compositions for the precursor ion type and eachfragment ion type of each retained triplet; (g6) estimating aprobability of the correctness of each respective estimated elementalcomposition estimated in step (f4); and (g7) calculating a probabilitythat the two fragment ion types were formed by fragmentation of theprecursor ion type of each retained triplet, based on the estimatedprobabilities of the correctness estimated elemental compositions.
 9. Amethod of tandem mass spectrometry as recited in claim 1, wherein themass spectrometer comprises an ion cyclotron resonance massspectrometers or an electrostatic trap mass spectrometer.
 10. A methodof tandem mass spectrometry as recited in claim 1, wherein the step (d)of generating a sample of fragment ions comprising plurality of fragmention types by fragmenting the plurality of precursor ion types within themass spectrometer comprises fragmenting the plurality of precursor ionssimultaneously.
 11. A method of tandem mass spectrometry as recited inclaim 1, wherein the step (b) of generating a mass spectrum of theprecursor ions and the step (d) of generating a mass spectrum of thefragment ion types are alternately performed by a single mass analyzerof the mass spectrometer.
 12. A method of tandem mass spectrometry asrecited in claim 11, wherein the step (d) of generating a sample offragment ions comprising plurality of fragment ion types is performed bya collision cell or reaction cell of the mass spectrometer such that theplurality of precursor ions are fragmented simultaneously.
 13. A methodof tandem mass spectrometry (MS/MS) for use in a mass spectrometercharacterized by the steps: (a) providing a sample of precursor ionscomprising a plurality of ion types, each ion type comprising arespective mass or range of masses; (b) generating a mass spectrum ofthe precursor ions using the mass spectrometer so as to determine arespective mass value or mass value range for each of the precursor iontypes; (c) estimating an elemental composition for each of the precursorion types based on the mass value or mass value range determined foreach respective ion type; (d) generating a sample of fragment ionscomprising plurality of fragment ion types by fragmenting the pluralityof precursor ion types within the mass spectrometer; (e) generating amass spectrum of the fragment ion types so as to determine a respectivemass value or mass value range for each respective fragment ion type;(f) estimating an elemental composition for each of the fragment iontypes based on the mass value or mass value range determined for eachrespective fragment ion type; and (g) calculating a set of probabilityvalues for each fragment ion type, each probability value representing aprobability that the fragment ion type was derived from a respective oneof the precursor ion types.
 14. A method of tandem mass spectrometry asrecited in claim 13, further characterized by the step: (h) generating asynthetic MS/MS spectrum for each respective fragment ion type based onthe calculated probability values.
 15. A method of tandem massspectrometry as recited in claim 14, further characterized by the step:(i) providing at least one of the synthetic MS/MS spectra as input to apeptide identification software product so as to identify a peptide.